The therapeutic strategies for treating loss of vision caused by retinal cell damage vary, but they are all directed to controlling the illness causing the damage, rather than reversing the damage caused by an illness by restoring or regenerating retinal cells. As one example, the treatments of uveitis are drawn from the knowledge of changes in the retinal environment when inflammation occurs. Corticosteroids, such as prednisone, are the preferred drug of treatment. However, these drugs are immunosuppressants with numerous side effects. As well, the systemic immunosuppression may have significant negative effects on the development of children as well as on adults in poor health, such as the elderly and patients with chronic disease. These patients must try alternative drugs such as alkylating agents or antimetabolites, which also have side effects. Clearly, patients with eye diseases remain vulnerable to sustaining permanent damage to the retinal cells, even if drug treatments are available. Thus, successful treatments of retinal cell damage will include approaches that aid in the regeneration of damaged retinal cells without causing the harmful side effects caused by current treatment methods.
A current area of study that will be important for treating diseases of the eye and other tissues involves the use of stem cells to regenerate damaged cells. Stem cells are undifferentiated cells that exist in many tissues of embryos and adult mammals. In embryos, blastocyst stem cells are the source of cells which differentiate to form the specialized tissues and organs of the developing fetus. In adults, specialized stem cells in individual tissues are the source of new cells which replace cells lost through cell death due to natural attrition, disease or injury. No stem cell is common to all tissues in adults. Rather, the term “stem cell” in adults describes different groups of cells in different tissues and organs with common characteristics.
Stem cells are capable of producing either new stem cells or cells called progenitor cells that differentiate to produce the specialized cells found in mammalian organs. Symmetric division occurs where one stem cell divides into two daughter stem cells. Asymmetric division occurs where one stem cell forms one new stem cell and one progenitor cell. A progenitor cell differentiates to produce the mature specialized cells of mammalian organs. In contrast, stem cells never terminally differentiate (i.e., they never differentiate into a specialized tissue cell). Progenitor cells and stem cells are referred to collectively as “precursor cells”. This term is used when it is unclear whether a researcher is dealing with stem cells or progenitor cells or both.
Progenitor cells may differentiate in a manner which is unipotential or multipotential. A unipotential progenitor cell is one which can form only one particular type of cell when it is terminally differentiated. A multipotential progenitor cell has the potential to differentiate to form more than one type of tissue cell. Which type of cell it ultimately becomes depends on conditions in the local environment such as the presence or absence of particular peptide growth factors, cell-cell communication, amino acids and steroids. For example, it has been determined that the hematopoietic stem cells of the bone marrow produce all of the mature lymphocytes and erythrocytes present in fetuses and adult mammals. There are several well-studied progenitor cells produced by these stem cells, including three unipotential and one multipotential tissue cell. The multipotential progenitor cell may divide to form one of several types of differentiated cells depending on which hormones act upon it.
FIG. 1 shows a schematic cross-section of the human eye, in which both the retina and the retinal pigment epithelium (RPE) are indicated. The retina is a layer of light sensitive tissue lining the inner surface of the eye. The RPE is a layer of pigmented cells just beneath the retina, which nourishes and supports the overlying retinal cells.
Retinal progenitor cells (RPCs) are multipotent, proliferative, and give rise to the various retinal cell types, while retinal pigment epithelium (RPE) progenitor cells normally generate solely RPE. Reh & Fischer, Methods Enzymol. (2006) 419:52-73. The RPE is a monolayer of neuroepithelial cells underlying and supporting the sensory retina. During development the RPE begins as a plastic tissue capable of regenerating lens or sensory retina but then differentiates very early [around E38 in humans and E9.5 in mouse (Bharti et al., Pigment Cell Res. (2006) 19(5):380-394)] and remains non-proliferative throughout life. RPE cells form a pigmented, single cell epithelium between the neural retina and the vascular choriocapillaris that has important roles in maintaining photoreceptor function. During development, the optic neuroepithelium evaginates into two outpocketings of the diencephalon. The dorsal aspect of the resulting optic vesicle is specified to generate the RPE, while the ventral aspect becomes neural retina. Bharti et al., Pigment Cell Res. (2006) 19(5):380-394. The other types of cells located in the retina include rod cells, cone cells, bipolar cells, amacrine cells, horizontal cells, Müller cells, glial cells, and retinal ganglion cells.
Interestingly, in amphibians, and in embryonic chick, RPE cells can produce other retinal and even lens tissues, indicating an inherent plasticity. Reh & Fischer, Methods Enzymol. (2006) 419:52-73. In amphibians, embryonic chick and embryonic rodents, RPE cells can proliferate and differentiate into neural progenitors and retinal cells. Id. This can occur in vivo and in vitro after induction with fibroblast growth factors (FGFs) (Park & Hollenberg, Dev. Biol. (1989) 134:201-205; Sakaguchi et al., Dev. Dyn. (1997) 209(4):387-398), or after enforced expression of retinal development genes, including Pax6, Ath5, NeuroD and NSCL, or after surgical removal of endogenous neural retina. Ma et al., Dev. Biol. (2004) 265(2):320-328; Azuma et al., Mol. Genet. (2005) 14(8):1059-1068; Yan & Wang, Neurosci. Lett. (2000) 280(2):83-86. In humans, RPE cells can proliferate in vivo, for example following retinal detachment (Machemer & Laqua, Am. J. Opthalmol. (1975) 80:1-23) and in vitro they proliferate and differentiate into β-tubulin III+ neurons, but their capacity to differentiate into a variety of neural cell types has not been demonstrated. Amemiya et al., Biochem. Biophys. Res. Commun. (2004) 316:1-5; Vinores et al., Exp. Eye Res. (1995) 60:385-400.
Retinal stem cells (RSCs) isolated from the ciliary epithelium and iris pigmented epithelium of adult rodents and humans have been described, and are reported to self-renew in vitro and differentiate into retinal neurons and glia. Reh & Fischer, Methods Enzymol. (2006) 419:52-73; Ohta et al., Dev. Growth Differ. (2008) 50:253-259. See also U.S. Pat. No. 6,117,675; and Tropepe et al., Science (2000) 287:2032-2036. However, these reports have been criticized by others, who have presented data indicating that the putative RSCs are, in fact, differentiated, pigmented ciliary epithelial cells. See Cicero et al., “Cells Previously Identified as Retinal Stem Cells Are Pigmented Ciliary Epithelial Cells,” Próc. Nat. Acad. Sci. USA. (e-publication Apr. 3, 2009) <URL http://www.pnas.org/cgi/doi/10.1073/pnas.0901596106> (last accessed Apr. 13, 2009). Moreover, there is as yet no evidence that a stem-like cell exists in the adult RPE. It is particularly important to look for the presence of these cells in humans, because of the relevance to human ocular diseases such as retinitis pigmentosa (RP), cone dystrophy, and age-related macular degeneration. da Cruz et al., Prog. Retin. Eye Res. (2007) 26:598-635.
There are no known successful treatments for RP and other retinal dystrophies. There are also no treatments which regenerate new cells endogenously or which transplant healthy tissue to the retina. Even if it were possible to develop some form of transplantation, it would be subject to the same problems that accompany transplants in other organ systems. These include: in many cases, implants provide only temporary relief as the symptoms associated with the disease often return after a number of years, rejection by the patient of foreign tissue, adverse reactions associated with immunosuppression (immunosuppression is needed to try to help the patient accept the foreign tissue), the inability of a sufficient number of cells in the tissue being implanted to survive during and after implantation, transmitting other diseases or disorders may be transmitted to the patient via the implant, and the results may not justify the costs and efforts of a complex procedure.
Thus, there is a need for new treatment options, other than transplantation, for the treatment of diseases of the retina and of many other tissues where cell regeneration would be beneficial. Given the immense potential for stem cells to provide new therapeutic treatments for a broad array of human diseases such as those described above, there remains a need for stem and/or progenitor cells that may be easily isolated from adult tissue, and that are multipotential, thereby having the capacity to differentiate into a broad array of different tissue types. The present invention describes such cells.
The citation and/or discussion of cited references in this section and throughout the specification is provided merely to clarify the description of the present invention and is not an admission that any such reference is “prior art” to the present invention. All cited references are incorporated herein by reference in their entirety.